Pancreatic cancers are particularly lethal and known for aggressive metastatic invasion. Over the prior funding period we have gathered substantial published and unpublished observations providing insight into how cells from these tumors disseminate. These findings support our CENTRAL HYPOTHESIS predicting that invasive pancreatic tumor cells and associated stromal cells in situ form a dynamic actin-based invadosome at the leading edge that selectively recruits specific metalloproteases to support adhesion, migration, and matrix remodeling during metastatic invasion. This hypothesis will test the contributions of the large polymeric GTPase Dynamin2 that forms a contractile scaffold linking the invadosome actin-based network to the advancing cell membrane. We will pursue three related but distinct specific aims that will provide mechanistic insights into the machinery, signaling, and degradative process utilized by invading PDAC cells and associated stroma. These aims include: One, defining the Dyn2/actin-based machinery that is essential for invadosome extension during migration in a 3-dimensional environment and in situ, with special emphasis on the expression, interaction, and function of the oncogenic actin adaptors, a-actinin 1 and 4, Two, understanding the regulation of invadosome dynamics by small oncogenic GTPases, that directly bind Dyn2, with an emphasis on a therapeutic approach, and Three, defining the mechanisms of matrix remodeling by the Dyn2- centric invadosome through the identification of novel pathways that regulate protease activity. This proposal will apply stat-of-the-art live-cell imaging methods to multiple experimental model systems that manipulate mixed populations of tumor and stromal cells in a 3D environment, as well as tumors resected from genetic mouse models and from human PDAC patients. The information gleaned from these approaches will greatly expand our mechanistic understanding of this disease while developing new therapies and treatments.